(1) Field of the Invention
The invention relates to the formation of immortalized cell lines and more particularly to immortalized epithelial cell lines infected with a hybrid SV40 adenovirus, as well as to methods of preparing the cell lines and uses therefore.
(2) Description of the Related Art
The establishment of lens epithelial cells in tissue culture is of considerable importance not only for the study of the problem of cell differentiation, but also is valuable for studying the etiology of primary and secondary cataract. It is also of interest as a progenitor of lens fibers in vivo and because information concerning the regulation of their growth and gene expression is limited. Although there have been a number of studies concerning cell lines of animal lens epithelial cells in culture, attempts to grow human lens epithelia have only been modestly successful.
It is well known that lens epithelial cells undergo a developmental transition into fiber cells of the lens cortex, a process characterized by distinct biochemical changes such as the synthesis of fiber-specific protein, .beta. and .gamma.-crystallins and morphological changes such as cell elongation, loss of cellular organelles and disintegration of the nucleus. The lens epithelium is located on the anterior surface of the lens immediately beneath the capsule and is thought to play a pivotal role in the development and progression of human cataracts, particularly those caused by exogenous mutagens. Data from experimentally induced cataracts and clinical experience suggest that primary damage to the genome of the epithelial cells, mediated by abnormal differentiation to lens fiber cells is collectively expressed as a cataract. See Worgul B. V., Merriam G. R. and Medvedovsky C. "Cortical cataract development--an expression of primary damage to the lens epithelium." Lens Eve Tox Res. 1989;6:559-571.
Early attempts to culture human lens epithelial cells were not very successful, due to their low proliferative ability in vitro, which decreased with age of the tissue. See, for example, Tassin J., Malaise E. and Courtois Y. "Human lens cells have an in vitro proliferative capacity inversely proportional to the donor age." Exp Cell Res. 1979; 123:388-392; Hamada Y. and Okada TS. "In vitro differentiation of cells of the lens epithelium of human fetus." Exp Eye Res. 1978; 26:91-97; Reddan J. R., McGee S. J., Goldenberg E. M., and Dziedzic. "Both human and newborn rabbit lens epithelial cells exhibit similar and limited growth properties in tissue culture." Curr Eye Res. 1982/1983; 2:399-405; and Jacob T. J. C., "Human lens epithelial cells in culture." Exp Eye Res. 1987; 45:93-104.
More recently, human lens epithelial cells have been cultured using fetal or infant lens epithelial explants. These efforts demonstrated the presence of crystallins but revealed their decreased production after several cell passages. See, Reddy V. N., Arita L. T., Zigler J. S., Jr. and Huang Q. L., "Crystallins and their synthesis in human lens epithelial cells in tissue culture." Exp Eye Res. 1988; 47:465-478; and Arita T., Lin L-R and Reddy V. N., "Differentiation of human lens epithelial cells in tissue culture." Exp Eye Res. 1988; 47:905-910.
Primary cultures of infant and fetal cells have been successfully grown and cultured through at least three (3) passages with the consistent formation of a monolayer with population doublings of up to 12 in culture. See Nagineni C. N., and Bhat S. P., "Human fetal epithelial cells in culture: an in vitro model for the study of crystallin expression and lens differentiation." Curr Eye Res. 1989; 8:285-291. These cells have been shown to express .alpha., .beta. and .gamma.-crystallins characteristic of epithelial cells undergoing differentiation into fibers, synthesize the capsule in vitro, and can undergo cell differentiation into fiber like cells of lentoids on low protein-binding surfaces or in co-cultures of lens epithelial cells and ciliary fibroblasts. Despite these advances, the practical utility of human lens epithelial cells is hindered by the limited availability of infant and fetal lenses, diminished in vitro growth after 4-6 population doubling levels (pdls) in cell culture, the lack of continuous cell lines, and the long proliferation times required to obtain a sufficient number of cells for use as a model for studying cell differentiation and the etiology of cataract.
Further studies have been undertaken with virus-transfection of mammalian cells with transforming virus to immortalize cells in vitro. Zeitlin PL., Lu L., Rhim J., Cutting G., Stetten G., Kieffer K. A., Craig R. and Guggino W. B., "A cystic fibrosis bronchial epithelial cell line: immortalization by Adeno-12-SV40 infection." Am. J. Respir. Cell Mol. Biol. 1991. 4:313-319; Hoffman M-C, Narisawa S., Hess R. A. and Millan J. L., "Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen." Exp Cell Res. 1992, 201:417-435; and Bartek J., Bartkova J., Kyprianou N., Lalani E-N., Staskova Z., Shearer M., Chang S. and Taylor-Papadimitrou J., "Efficient immortalization of luminal epithelial cells from mammary gland by introduction of similar virus 40 large tumor antigen with a recombinant retrovirus." Proc. Natl. Acad. Sci. USA 1991; 88:3520-3524. Limited success has been reported with bovine and rat lens epithelium. Miller G. G., Blair D. G., Hunter E., Mousa G. Y. and Trevithick J. R., "Differentiation of rat lens epithelial cells in tissue culture (III). Functions in vitro of a transformed rat lens epithelial cells line". Develop Growth an Differ. 1979;21:19-27. Bovine lens epithelial cells have also been conditionally immortalized by a temperature-sensitive mutant; of the SV40 virus and rat lens epithelial cells have been transformed using rous sarcoma virus, retaining partially differentiating function as shown by .beta.-crystallin synthesis. No immortalized cell lines exist, however, for the human lens epithelium. However, the availability of such a cell line would greatly enhance the study of human lens epithelial physiology, and may aid in the design of drugs that inhibit both primary and secondary human cataracts. Thus, the availability of a cell line which maintains the normal differentiating functions of the lens epithelial cells would be of practical importance in the study of cataractogenesis and agents that inhibit cataract.